The purpose of the proposed study is to elucidate the molecular mechanism of catecholamine stimulated non-shivering thermogenesis. Both skeletal muscle and brown adipose tissue have been implicated as largely responsible for the 2-3 fold increased in O2 consumption observed in cold-adapted rats given catecholamines or exposed to low ambient temperatures. In the present study the perfused rat hemicorpus will be used to study skeletal muscle metabolism and isolated brown fat adipocytes to study the brown adipose tissue metabolism. Rats will be cold-adapted by exposing them to 4 degrees C ambient temperatures for 3-4 weeks prior to use. An initial goal was to determine whether increased rates of tissue respiration which occur in the presence of norepinephrine of isoproterenol result from increased substrate delivery, increases in cytosolic ADP due to increases in sodium pump activity or uncoupling of mitochondrial O2 uptake from phosphorylation. This goal has been partially realized in the first year by comparing rates of O2 uptake of mitochondria in the cell with those of isolated mitochondria. Properly prepared isolated mitochondria respire as rapidly as mitochondria in the tissue only when uncoupled. Further approaches used will be 1) to measure sodium pump activity isotopically by loading the tissue with 22Na ion and then measuring efflux rates and 2) to vary substrate delivery to the tissues by artificial means. The study is unique and may provide some new perspectives on the mechanisms of non-shivering thermogenesis because it employs an adequate model of muscle perfusion. The studies of brown fat are likewise unique because new techniques of rapid cell fractionation will be used to determine compartmentation of critical metabolites between cytosol and mitochondria.